Post-translational modifications by ubiquitin-like (Ubl) family proteins play essential roles in many cellular processes. Two Ubls, ubiquitin and SUMO, have extensive involvement in maintaining genome integrity. Evolutionarily conserved SUMO-targeted E3 ubiquitin ligases (STUbLs) connect the SUMO and ubiquitin pathways by targeting sumoylated proteins for ubiquitylation and proteasomal degradation. STUbLs modulate SUMO pathway homeostasis, and are connected to genome stability and tumorigenesis. Despite their functional importance, the molecular mechanism and physiological substrates of STUbLs remain largely undefined. The three Specific Aims in this proposal focus on elucidating the molecular mechanism of STUbL function in genome maintenance. In Aim 1, STUbL in vivo substrates and interacting protein networks will be identified by mass spectrometry-based proteomic analyses. In Aim 2, the mechanism of regulation on genome stability by STUbLs will be examined. The effect of STUbL activity on DNA damage response (DDR) and whether STUbLs target specific DDR factors will be investigated. In Aim 3, several genetic screens will be employed to comprehensively define STUbL functions. To achieve the above aims, a combination of powerful yeast genetics, mass spectrometry, living microscopy, molecular biology, and biochemistry tools will be employed. The observations made in the proposed studies will provide key insights into the mechanism by which STUbLs safeguard genome stability as well regulate other cellular functions. Furthermore, information derived from this research may form the basis of therapeutic strategies for treating human diseases evolved from genome damage and instability. PUBLIC HEALTH RELEVANCE: A new family of proteins, STUbLs, is key to maintaining genome stability and cancer suppression. Studies in this proposal focus on understanding the mechanisms and functions of STUbLs, and will contribute to therapeutic strategies for treating human diseases.